Comparative Study Of Dna Preservation Under Various Conditions On Local Egyptian Cowpea Germplasm

This study were to study the impact of storage conditions on DNA quality in different materials, the integrity and quality of stored DNA; as well as determining the best way to store of DNA extracts in some Cowpea plants collected from different sites in Egypt. Ten germplasm representing Vigna radiata and V. unguiculata, procured from local regions by National Gene Bank (NGB), Agricultural Research Centre (ARC), Giza, Egypt. The DNA from each treatment was stored in different buffers for at least a year at LN, –20°C, –80°C and room temperature (RT) without special control of temperature or humidity. In order to simulate and evaluate the long-term storage samples subjected to high temperature over a period of time by extrapolation could correspond to 100 years at 25°C. These ex periments were conducted on the samples of ten samples of Vigna at 65°C for one day in hot-plate. The obtained results, the protectant trehalose were the highest value of DNA concentration (43.6±1.1 ng/µL) at room temperature, following by PVA scored 43.5±1.0 ng/ µL at RT and TE buffer showed 43.5±0.7 ng/µL at -20°C, for stored DNA. The average mean of PVA and trehalose were 43.5±1.1 ng/ µL more than TE-buffer. For stored tissue, the high mean (43.5±0.8 ng/µL) value of DNA concentration scored with the silica-gel and herbarium specimens, respectively. DNA samples of ten samples under study were also subjected to PCR analysis using SSR to determine whether the DNA was degraded. While no degradation was observed for DNA stored under different conditions, special in dry conditions (≤ 5% humidity). To assess its effects on DNA stability during storage at different conditions under controlled humidity. It was very interesting to note that DNA samples stored in the presence or absence of protectant additives exhibited no detectable degradation, whereas in the absent of, DNA was degraded and appeared as a smear on an agarose gel.


Introduction ISSN 2471-6782
This study were to study the impact of storage conditions on DNA quality in different materials, the integrity and quality of stored DNA; as well as determining the best way to store of DNA extracts in some Cowpea plants collected from different sites in Egypt. Ten germplasm representing Vigna radiata and V. unguiculata, procured from local regions by National Gene Bank (NGB), Agricultural Research Centre (ARC), Giza, Egypt. The DNA from each treatment was stored in different buffers for at least a year at LN, -20°C, -80°C and room temperature (RT) without special control of temperature or humidity. In order to simulate and evaluate the long-term storage samples subjected to high temperature over a period of time by extrapolation could correspond to 100 years at 25°C. These experiments were conducted on the samples of ten samples of Vigna at 65°C for one day in hot-plate. The obtained results, the protectant trehalose were the highest value of DNA concentration (43.6±1.1 ng/µL) at room temperature, following by PVA scored 43.5±1.0 ng/ µL at RT and TE buffer showed 43.5±0.7 ng/µL at -20°C, for stored DNA. The average mean of PVA and trehalose were 43.5±1.1 ng/ µL more than TE-buffer. For stored tissue, the high mean (43.5±0.8 ng/µL) value of DNA concentration scored with the silica-gel and herbarium specimens, respectively. DNA samples of ten samples under study were also subjected to PCR analysis using SSR to determine whether the DNA was degraded. While no degradation was observed for DNA stored under different conditions, special in dry conditions (≤ 5% humidity). To assess its effects on DNA stability during storage at different conditions under controlled humidity. It was very interesting to note that DNA samples stored in the presence or absence of protectant additives exhibited no detectable degradation, whereas in the absent of, DNA was degraded and appeared as a smear on an agarose gel.
Biodiversity is very important for conserving genetic diversity present in and available to our current and potential crop species. Cultivated crops are extremely inbred for factors like yield, uniform flowering and height, and cosmetic features of products. This narrow genetic base has resulted in several disastrous crop failures (Adams, 1997). It is well known that there are two approaches to conservation of PGR -ex situ and in situ. Ex situ conservation approach generally comprises the following methods: seed storage, field gene-bank, in vitro storage, pollen storage, DNA storage and botanical gardens. The genomic DNA extraction is a primordial stage in several practices and genetic analyses such as those that involves the use of molecular markers, which has had a growing application in the plant systematics and in the population analysis. Studies based in molecular markers can be used successfully in phylogenetic analyses, serving as a great help in matters unresolved by traditional  , 1993). However, in studies that involve the collection of wild plants it is not always possible, since in most cases the populations are far from the research laboratory. An alternative is to perform the freezing of plant material in liquid nitrogen during collection in the field. However, this practice is not feasible in many cases, given the difficulty and danger of transporting liquid nitrogen container in rough terrain and difficult access. Thus, a method much used in the preservation of plant tissue for subsequent DNA extraction is rapid dehydration in silica gel (Witono and Kondo, 2006;Whitlock et al., 2008). This technique is generally simple and efficient, since the dehydrated state, the DNA is less susceptible to chemical or enzymatic degradation (Murray and Thompson, 1980). However, some species do not respond well to this type of conservation, with losses in quality of the obtained DNA. Other ways of preserving material may be used; however, not all are simple or require the availability of some specific equip-

Materials and Methods
Ten germplasm representing Vigna radiata and V. unguiculata subspecies cv-group: unguiculata, procured from local regions by National Gene Bank (NGB), Agricultural Research Centre (ARC), Giza, Egypt. The studied accessions of genus Vigna, collected date and area were presented in Table (1). ment such as freeze dryers. The progress in genetic engineering has resulted in breaking down the species and genus barriers for transferring genes (National Research Council, 1993). Transgenic plants have been produced with genes transferred from viruses, bacteria, fungi and even mice. Such efforts have led to the establishment of DNA libraries, which store total genomic information of germplasm (Mattick et al., 1992). However, strategies and procedures have to be developed on how to use the material stored in the form of DNA. Therefore, the role and value of this method for PGR conservation are not completely clear yet (Adams et al., 1992). This study therefore were to study the impact of storage conditions on DNA quality in different materials, the integrity and quality of stored DNA; as well as determining the best way to store of DNA extracts in some Cowpea plants collected from different sites in Egypt.

Statistical analysis
Total genomic DNA from 5 g of leaf tissue per germplasm was extracted following the Zymo Research. Electrophoresis was made on 1% agarose gel electrophoresis at 100 Volt for 30 min. The total genomic DNA was diluted to 10 ng/μl for PCR analysis. The herbarium voucher-specimens are deposited at the herbarium Department of Taxonomy, National Gene Bank (NGB. While, extracted gDNA was used to test different treatments and storage conditions. The used protective-agents applied to add with DNA samples trehalose (5µL of 10%), according to Taylor  The herbarium voucher-specimens are deposited at the herbarium Department of Taxonomy, National Gene Bank (NGB. While, Ten microsatellite primers (Table 2) were evaluated to amplify the success PCR after the storage. The PCR reactions were performed in a 20 μl reaction mixture containing 10 ng template DNA, 200 μM dNTPs, 250 nM of each primer, 1.5 mM MgCl2, 1x PCR buffer and 1 unit Taq DNA polymerase. The PCR amplification was performed with an initial denaturation at 94°C for 5 min followed by 35 cycles of 94°C for 1 min, 53 to 60°C (depending on the primer) for 1 min, 72°C for 2 min, and a final extension at 72°C for 5 min before cooling at 4°C.
Basing on the averages of all attributes, data set were fed into SPSS (version. 14.0) and StatistiXL adding in Microsoft Excel (Kovach Computing Service 2013, version. 1.8) Program. extracted gDNA was used to test different treatments and storage conditions.
The results can be represented as an interaction of several independent factors affecting DNA preservation, such as concentration, protective agent, and temperature, represented onto Fig. 1. The DNA from each treatment was stored in different buffers for at least a year at LN, -20°C, -80°C and room temperature (RT) without special control of temperature or humidity. In order to investigate whether the storage time affected sample recovery and rehydrated samples of DNA were analyzed using specific microsatellite primers, see Table 2. In order to simulate and evaluate the long-term storage samples subjected to high temperature over a period of time by extrapolation could correspond to 100 years at 25°C. These experiments were conducted on the samples of ten germplasm of Vigna at 65°C for one day in hot-plate. The fluorometric analysis indicated that the overall there was little or no difference in the amount of DNA recovered as compared to the control samples. The obtained results are shown in Table  3. The obtained results, the protectant trehalose were the highest value of DNA concentration (43.6±1.1 ng/µL) at room temperature, following by PVA scored 43.5±1.0 ng/µL at RT and TE buffer showed 43.5±0.7 ng/µL at -20°C, for stored DNA. On the other hand, the average mean of PVA and trehalose were 43.5±1.1 ng/µL more than TE-buffer. For stored tissue, the high mean (43.5±0.8 ng/µL) value of DNA concentration scored with the silica-gel and herbarium specimens, respectively. In general, the control appeared the highest value of DNA concentration (43.7±1.1 ng/µL) comparing with the whole treatments (Fig. 1); following by the value of trehalose (43.6±1.1 ng/µL). After a month of storage at a different degree of temperatures, no degradation was seen whereas, as shown in Fig. (1), no visible differences of the samples occurred after when compared to the control sample. As any cut in the target sequence will prevent its amplification, DNA samples of ten accessions under study were also subjected to PCR analysis using SSR (using primers: EH-05, EH-02 and EH-07, see Table 2 for more details) to determine whether the DNA was degraded as shown in Figure 2. While no degradation was observed for DNA stored under different conditions, special in dry conditions (≤ 5% humidity). To assess its effects on DNA stability during storage at different conditions under controlled humidity. It was very interesting to note that DNA samples stored in the presence or absent of protectant additives exhibited no detectable degradation, whereas in the absent of, DNA was degraded and appeared as a smear on an agarose gel (Figure 2). From the results obtained, the best way to protect DNA during storage was treated with trehalose. These results agreed with Ivanova and Kuzmina (2013) and Clermont et al. (2014). The results presented demonstrate the properties of protective be not affected by storage extreme conditions but guaranteeing for long term without degradation of DNA as well as the stored DNA can be used in downstream applications such as PCR.  (Jones et al., 2007). Among the most commonly used disaccharides (sucrose and trehalose), trehalose is preferable for stabi-lization of biomolecules due to its higher transition temperature. It stabilizes DNA due to its ability to form tight hydrogen bonds to the phosphate groups, which leads to shielding of the large phosphate-phosphate repulsion. As well as, it probably interacts with other polar groups of DNA that makes trehalose a water-like solvent for DNA and stabilizes the bas stacking during and after dehydration (Zhu et al., 2007). The advantages of trehalose can be summarized as follows: more flexible formation of hydrogen bonds with proteins and DNA due to the absence of internal hydrogen bonds; less hygroscopicity; low chemical reactivity; and prevention of water plasticizing the amorphous phase partly by forming trehalose-protein-water microcrystals (Crowe et al., 1992;librizzi et al., 1999). PVA is a polymer which has many desirable characteristics specifically for various applications (Hassan et al., 2000). It can be successfully used for PCR typing analysis (Schyma et al., 1999). The studies of PVA-DNA cryogel have demonstrated strong interaction between DNA and PVA (Papancea et al., 2008). The hydrogen bonding between DNA and PVA enables preparation of PVA-DNA nanoparticles suitable for gene and protein delivary applications (Liao et al., 2005). Besides, this compound is not toxic, does not affect enzymatic reactions, and keeps some amount of water molecules tightly attached even in dehydrative solvent which is advantageous for enzyme stabilization (Szczęzsna-Antczak et al., 2002). Figure (2)The banding profile detected by SSR primer for ten samples stored in different buffers at -20°C, -80°C and RT.